Plant Molecular Biology 29: 267-278, 1995. © 1995 Kluwer Academic Publishers. Printed in Belgium. 267 Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii Adrian P. Brown, Clare L. Brough, Johan T. M. Kroon and Antoni R. Slabas Department of Biological Sciences, University of Durham, South Road, Durham DHI 3LE, UK Received 14 March 1995; accepted in revised form 12 July 1995 Key words: 1-acyl-sn-glycerol-3-phosphate acyltransferase, complementation cloning, Limnanthes douglasii Abstract Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3- phosphate acyltransferase (LPA-AT) enzymes from Limnanthes douglasii. Both heterologous screening with the maize pMAT1 clone and in vivo complementation of the Escherichia coli mutant JC201 which is deficient in LPA-AT activity, were carried out. Clones identified by these procedures were different. Homology searches demonstrated that the clone isolated by heterologous probing, pLAT1, encodes a protein which is most similar to the maize (open reading frame in pMAT1) and yeast SLC1 proteins, which are putative LPA-AT sequences. This L. douglasii sequence shows much lower homology to the E. coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies. The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PIsC. It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a substrate specificity distinctly different from PlsC and similar to Limnanthes sp. microsome specificity. This data strongly supports the contention that pLAT2 is an LPA-AT clone. Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2. Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A) + mRNA from seed, stem and leaf, demonstrating more widespread expression throughout the plant. Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small gene family. Introduction Modification of naturally occurring triacylglycer- ols (TAGs) in oilseed crops to provide industri- ally useful products is currently of great interest. One aim is the development of a Brassica napus cultivar which is able to incorporate very high levels of erucic acid (22:1 A13) into its seed oil and preferably synthesize large amounts of trieru- cin (trierucoglycerol) [25]. Currently available The nucleotide sequence data reported will appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession numbers Z46836 (pLAT2) and Z48730 (pLAT1).